www.nature.com/scientificreports OPEN The novel Dbl homology/ BAR domain protein, MsgA, of Talaromyces marnefei regulates yeast morphogenesis during growth inside host cells Harshini Weerasinghe1,2, Hayley E. Bugeja1 & Alex Andrianopoulos1* Microbial pathogens have evolved many strategies to evade recognition by the host immune system, including the use of phagocytic cells as a niche within which to proliferate. Dimorphic pathogenic fungi employ an induced morphogenetic transition, switching from multicellular hyphae to unicellular yeast that are more compatible with intracellular growth. A switch to mammalian host body temperature (37 °C) is a key trigger for the dimorphic switch. This study describes a novel gene, msgA, from the dimorphic fungal pathogen Talaromyces marnefei that controls cell morphology in response to host cues rather than temperature. The msgA gene is upregulated during murine macrophage infection, and deletion results in aberrant yeast morphology solely during growth inside macrophages. MsgA contains a Dbl homology domain, and a Bin, Amphiphysin, Rvs (BAR) domain instead of a Plekstrin homology domain typically associated with guanine nucleotide exchange factors (GEFs). The BAR domain is crucial in maintaining yeast morphology and cellular localisation during infection. The data suggests that MsgA does not act as a canonical GEF during macrophage infection and identifes a temperature independent pathway in T. marnefei that controls intracellular yeast morphogenesis. Host defence against disease causing microbes involves actively identifying and eliminating invading pathogens. Tis begins with the innate immune system where host phagocytes engulf and destroy these microbes, followed by triggering of an adaptive immune response. Te success of a pathogen depends on its ability to escape the host response and for some pathogens this involves residing within particular phagocytic cells of the host where they are then able grow and proliferate. An important factor facilitating this lifestyle is the capacity to adopt a growth form suitable for the spatial constraints of the intracellular environment of a host cell. For many fungal pathogens that utilise the intracellular environment of macrophages as a means of immune system avoidance, morphological plasticity is an important virulence attribute 1–6. Tis is exemplifed by the dimorphic fungi, which display saprophytic, multicellular, flamentous hyphal growth in the external environment and are able to adopt a unicellular yeast growth form during infection. Dysregulation of morphology, leading to the production of a growth form that disrupts the integrity of the phagocytic cells, exposes the pathogen to the entire immune system. Talaromyces marnefei (formerly Penicillium marnefei) is an intracellular human pathogen that exhibits temperature dependent dimorphic growth. At 25 °C it grows in a multinucleate septate hyphal form that can diferentiate to produce uninucleate asexual spores (conidia)7,8. At 37 °C in vitro it grows as uninucleate yeast that divides by fssion. Te transition from hyphal cell or dormant conidium to a yeast cells proceeds by arthro- conidiation, a process of flament fragmentation, in which nuclear and cellular division are coupled, double septa are laid down with subsequent cell separation producing yeast cells8. Te transition from yeast cell to hyphal cell occurs by polarised growth at the tips of the elongate yeast cells and a switch to cell division by septation without subsequent cell separation. Te conidia are the infectious propagules and infection is believed to be initiated through their inhalation 9. Once within the human host, T. marnefei conidia are engulfed by host primary alveolar macrophages where they bypass the process of arthroconidiation and germinate directly into yeast cells, which are the pathogenic 1Genetics, Genomics and Systems Biology, School of BioSciences, University of Melbourne, Victoria 3010, Australia. 2Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton 3800, Australia. *email: [email protected] Scientifc Reports | (2021) 11:2334 | https://doi.org/10.1038/s41598-020-79593-4 1 Vol.:(0123456789) www.nature.com/scientificreports/ form. While hyphae and yeast are the predominant morphologies for T. marnefei, particular growth conditions manifest distinct diferences in cell shape and length for some cell types. For instance, in vitro grown T. marnefei yeast cells have an elongated, flament-like morphology, whilst in vivo produced yeast cells are short, ellipsoid and compact, a form more suited for growing within the confnes of the macrophage. As an intracellular pathogen the ability of T. marnefei to tightly regulate the dimorphic switch and maintain yeast morphology in the host is crucial for pathogenicity. Terefore, temperature drives the dimorphic switch and host signals modify yeast cell morphogenesis. In a number of animal and plant pathogenic fungal species, cell type specifc morphogenesis is controlled by signalling pathways involving the small GTPase molecular switches and their accessory factors; guanine- nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) (reviewed in10). Tese play crucial roles in processes associated with cell shape such as control of polarized growth and cytoskeletal organisation. Rho-type GEFs are responsible for activating their small GTPase targets and their activity has been shown to regulate cytokinesis, cell wall integrity, antifungal resistance, viability, tissue invasive growth, mycotoxin pro- duction and virulence 11–14. Te majority of these studies have focused on GEFs containing canonical structural domains—a catalytic Dbl homology domain (DH) domain and an auxiliary plekstrin homology (PH) domain, excluding the contribution of GEFs with alternate domain structures (reviewed in15). In T. marnefei, a number of small GTPases of the Ras superfamily and associated downstream efectors have been shown to infuence morphogenesis of all cell types in specifc and distinct patterns, during growth under both in vitro and ex vivo (intracellular macrophage) growth conditions, by afecting polarity establishment and the diferentiation of distinct cell types16. For example, the Cdc42 orthologue in T. marnefei, encoded by cfA, is required for correct yeast and hyphal cell morphogenesis in vitro, as well as germination of conidia but does not afect the various cell types or diferentiation of asexual development structures (conidiophores). Whereas the Rac orthologue, encoded by cfB, is important for hyphal cell morphogenesis and asexual development but not yeast cell morphogenesis17,18. In addition, orthologues of the p21 activated kinases (PAK) Ste20 and Cla4, encoded by pakA and pakB respectively, are important for conidial germination and yeast cell morphogenesis at 37 °C19,20. In particular pakB exclusively afects the formation of yeast cells during intracellular growth, sug- gesting that the pathways controlling morphogenesis of T. marnefei in vitro and during intracellular growth in host cells have some unique components that respond to distinct cues. In a T. marnefei expression profling study examining genes expressed during growth inside macrophages, a gene was identifed that was specifcally upregulated inside murine macrophages and was predicted to encode a RhoGEF-like protein (Weerasinghe et al. 2018 in prep). Tis previously uncharacterised gene that was named msgA (macrophage specifc GEF-like A) is the only RhoGEF-like encoding gene in T. marnefei to show this specifc pattern of expression. While MsgA contains a DH domain, unlike canonical GEFs, a Bin-Amphiphysin- Rvs (BAR) domain replaces the PH domain. Proteins containing BAR domain have been shown to play various roles in membrane dynamics as well as interacting with small GTPases and many other proteins21–24. Deletion of msgA results in aberrant yeast morphology during macrophage infection but not during in vitro growth at 37 °C. Induced overexpression of msgA during in vitro growth resulted in yeast cell formation mimicking that of growth inside macrophages. Mutational analysis showed that the BAR domain of MsgA is crucial in establishing correct yeast morphogenesis and localisation during intracellular growth. Together these results defne a novel host infection specifc pathway that regulates intracellular morphogenesis in T. marnefei. Materials and methods Molecular methods and plasmid construction. T. marnefei genomic DNA was isolated as previously described25. Southern and northern blotting was performed using Amersham Hybond N+ membrane and [a-32P] dATP labelled probes by standard methods26. Sequences of primers are provided in Supplementary Table S1. Deletion constructs were created using a modifed Gateway™ method27. Te deletion construct of msgA was created using pHW7711 containing the pDONR-pyrG cassette. Wildtype PCR product of msgA (PMAA_089500) was generated with primers VV55 and VV56 and cloned into pBluescript II SK+ to generate pHW7897. To make the deletion construct, pHW7905, PCR primers VV57 and VV58 were used to generate an inverse PCR product for the Gateway™ reaction. To create a complementation construct, wildtype PCR product of msgA was cloned into the niaD targetting plasmid pHB7615 to generate pHW8053. Te overexpression allele xylP(p)::msgA (pHW8056) was generated by PCR amplifcation of 2 kb of the 5′ promoter and ORF regions of the msgA gene using WW78 and WW77 to create subclone pHW8069. An inverse PCR product of pHW8069